Metallohydrolase biomimetics with catalytic and structural flexibility

Article abstract of DOI:10.1039/c6dt03200a

The structural and functional properties of zinc(ii) complexes of two nitrogen rich polydentate ligands, HTPDP = 1,3-bis(bis-pyridin-2-ylmethylamino)propan-2-ol and HTPPNOL = N,N,N′-tris-(2-pyridylmethyl)-1,3-diaminopropan-2-ol, are compared. HTPDP is a hepta-dentate ligand with four pyridyl groups attached to a 1,3-diaminopropan-2-ol backbone while HTPPNOL contains only three pyridyl groups. In reactions with Zn(ClO₄)₂, HTPDP forms a dinuclear zinc compound [Zn₂(TPDP)(OAc)](ClO₄)₂, 1. On the other hand, mononuclear [Zn(HTPPNOL)](ClO₄)₂, 2, and tetranuclear [Zn₄(TPPNOL)₂(OAc)₃](ClO₄)₃, 3, complexes were isolated with the ligand HTPPNOL. Kinetic measurements with the substrate bis(2,4-dinitrophenyl)phosphate (BDNPP) revealed that compound 1 (k_cat = 31.4 × 10^?3 min^?1) is more reactive than 3 (k_cat = 7.7 × 10^?3 min^?1) at pH = 8.5, whilst the mononuclear compound 2 is inactive. Compound 1 displays a typical steady-state kinetic behaviour, while compound 3 exhibits steady-state behaviour only ～120 s after starting the reaction, preceded by a burst-phase. ³¹P NMR studies confirm that 1 can promote the hydrolysis of both ester bonds in BDNPP, generating the monoester DNPP and inorganic phosphate in the process. In contrast, DNPP is not a substrate for 3. The crystal structure of the complex formed by 3 and DNPP reveals the formation of a tetranuclear zinc complex [Zn₄(TPPNOL)₂(DNPP)₂](ClO₄)₂, 4, in which the phosphate moiety of DNPP adopts an unusual tridentate μ-η¹:η¹:η¹ coordination mode.

Full text of DOI:10.1039/c6dt03200a

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Metallohydrolase biomimetics with catalytic and
structural flexibility†
Cite this: DOI: 10.1039/c6dt03200a
Luisa L. Mendes,a Daniel Englert,b Christiane Fernandes,a Lawrence R. Gahan,c
Gerhard Schenk*c and Adolfo Horn, Jr.*a
The structural and functional properties of zinc(II) complexes of two nitrogen rich polydentate ligands,
HTPDP
= 1,3-bis(bis-pyridin-2-ylmethylamino)propan-2-ol and HTPPNOL = N,N,N’-tris-(2-pyridyl-
methyl)-1,3-diaminopropan-2-ol, are compared. HTPDP is a hepta-dentate ligand with four pyridyl
groups attached to a 1,3-diaminopropan-2-ol backbone while HTPPNOL contains only three pyridyl
groups. In reactions with Zn(ClO4)2, HTPDP forms a dinuclear zinc compound [Zn2(TPDP)(OAc)](ClO4)2, 1.
On the other hand, mononuclear [Zn(HTPPNOL)](ClO4)2, 2, and tetranuclear [Zn4(TPPNOL)2(OAc)3]
(ClO4)3, 3, complexes were isolated with the ligand HTPPNOL. Kinetic measurements with the substrate
bis(2,4-dinitrophenyl)phosphate (BDNPP) revealed that compound 1 (kcat = 31.4 × 10−3 min−1) is more
reactive than 3 (kcat = 7.7 × 10−3 min−1) at pH = 8.5, whilst the mononuclear compound 2 is inactive.
Compound 1 displays a typical steady-state kinetic behaviour, while compound 3 exhibits steady-state
behaviour only ∼120 s after starting the reaction, preceded by a burst-phase. 31P NMR studies confirm
that 1 can promote the hydrolysis of both ester bonds in BDNPP, generating the monoester DNPP and in-
organic phosphate in the process. In contrast, DNPP is not a substrate for 3. The crystal structure of the
Received 11th August 2016,
Accepted 26th September 2016
complex formed by
3 and DNPP reveals the formation of a tetranuclear zinc complex
DOI: 10.1039/c6dt03200a
[Zn4(TPPNOL)2(DNPP)2](ClO4)2, 4, in which the phosphate moiety of DNPP adopts an unusual tridentate
μ–η1:η1:η1 coordination mode.
www.rsc.org/dalton
possess a heterovalent binuclear centre with an iron(III) and a
second divalent metal ion, generally a zinc(II) or manganese(II)
Introduction
Metallohydrolases are a large and diverse family of enzymes for plant enzymes and a redox-active iron for their mammalian
that cover a broad range of metabolic functions, including counterparts.15–17 Metallophosphatases such as PAPs have
DNA proofreading, biosynthesis of a range of biomolecules, received considerable attention not only due to their mechan-
and also the control of neurological functions.1,2 Some of istic diversity but also due to the broad spectrum of substrates
these enzymes recently evolved specific activities to address they are capable of hydrolyzing. Such enzymes have been
environmental and medical challenges such as the increased shown to act on not only mono, di- and tri-phosphate esters,
use of pesticides and antibiotics.3–5 Whilst carboxypeptidase including natural substrates such as carbohydrates, peptides,
A6,7 and thermolysin8–10 are examples of mononuclear metallo- DNA and RNA, but also on synthetic compounds such as pesti-
hydrolases most metallohydrolases require a set of two identi- cides and nerve gases.4,18–27
cal divalent ions like zinc(II), nickel(II) or manganese(II).11–14
The development of synthetic analogs for such metallopho-
An exception are purple acid phosphatases (PAPs), which sphatases has provided useful benchmarks to address ques-
tions about the structure and mechanism of such
enzymes.1,28,29 Furthermore, such compounds also hold sig-
aLaboratório de Ciências Químicas, Universidade Estadual do Norte Fluminense,
nificant potential for various applications, including as che-
28013-602 Campos dos Goytacazes/RJ, Brazil. E-mail: adolfo@uenf.br;
motherapeutics in anticancer treatments30–32 or catalysts for
Fax: +55 22 2739 7046; Tel: +55 22 2739 7384
the degradation of toxic organophosphates.4,26,33,34 One of the
most successful families of ligands employed in the synthesis
of metallophosphatase models has been built on the 2,6-bis
(aminomethyl)-4-methylphenol backbone, which may provide
as many as seven ligands for the coordination environments of
the two metal ions, and in which a cresol unit acts as the
metal-bridging group.35,36 Both symmetric and non-symmetric
bAnorganisch-Chemisches Institut, Universität Heidelberg, Im Neuenheimer Feld 270,
69120 Heidelberg, Germany
cSchool of Chemistry and Molecular Biosciences, The University of Queensland,
Brisbane, QLD 4072, Australia. E-mail: schenk@uq.edu.au; Fax: +61 7 3365 4273;
Tel: +61 7 3365 4144
†Electronic supplementary information (ESI) available. CCDC 1495702–1495704.
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c6dt03200a
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Fig. 1 Scheme showing the structure of the ligands and complexes prepared in this work and the phosphate esters BDNPP and DNPP as well.
examples have been described.37–42 Features such as substitu- the properties of zinc(II) complexes generated with either a
ent effects,43 bridging groups32,36,44,45 and, more recently, the hepta- (HL1) or hexa- (HL2) dentate ligand (Fig. 1).
effect of the second coordination sphere have been evaluated
for their influence on the catalytic performance of such
biomimetics.44,46–48 Depending on the metal ion employed in
Experimental
the synthesis, the addition of carboxylate groups (e.g. acetate,
General methods
benzoate) to the reaction mixture may result in five- or six-co-
1H and 13C NMR (see the ESI†) spectra were recorded with a
ordinated metal ions. In an aqueous environment such carbox-
300, 400 or 500 MHz Bruker Advance Spectrometer. Data pro-
cessing was carried out with TOPSPIN from Bruker. Chemical
shifts of the final ligands were assigned using two-dimen-
sional correlation spectroscopy (COSY), heterodinuclear single
quantum correlation (HSQC), heterodinuclear multiple bond
connectivity (HMBC) and distortionless enhancement by polar-
ization transfer (DEPT). Infrared spectra were recorded using a
PerkinElmer FT-IR/NIR Frontier Spectrometer with a diamond/
ZnSe crystal, using a Universal ATR Sampling Accessory.
Elemental analysis for carbon, hydrogen and nitrogen was
carried out at the University of Queensland by Mr George
Blazak or Dr Michael Nefedov.
ylate ligands may be displaced by water molecules, resulting in
the coordination of water molecules to the metal ions, which,
at suitable pH values, may form hydroxo species that play an
essential role in the reactivity of these biomimetics.49–57
Another dinucleating unit that has been employed in the syn-
thesis of metallophosphatase models is the 1,3-diamino-2-
hydroxypropane molecule,58,59 which also provides up to seven
metal-coordinating ligands, and with a deprotonated alcohol
group acting frequently as a metal-bridging unit. For this
ligand system symmetric compounds are easily synthesized,
but their non-symmetric counterparts require more elaborate
synthetic pathways.60,61 This may also be one of the main
reasons why the majority of synthetic models for phosphatase
use aromatic rather than aliphatic bridging ligands.
X-ray crystallography
Although the effects of the substituent on the aromatic Crystallographic measurements were carried out using an
rings and the importance of the hydrogen donor groups for Oxford Diffraction Gemini Ultra dual source (Mo, λKα
=
the catalytic reaction have been described, a point that has 0.71073 Å and Cu, λKα = 1.5418 Å) CCD diffractometer. Crystal
been scarcely addressed is related to the presence of an structures were solved by either direct methods (SIR-92) or
additional vacant site in the first coordination sphere of the Patterson’s method (SHELX 86) and refined (SHELXL 97) by
dinuclear metal centre, mainly present with ligands that full matrix least squares methods.62 These programs were
contain the 1,3-diamino-2-hydroxypropane unit. Specifically, accessed through the WINGX 1.70.01 crystallographic collec-
the effect of an additional vacancy in the coordination environ- tive package.63 All non-hydrogen atoms were refined anisotro-
ment on the catalytic proficiency of such biomimetics is pically unless they were disordered. Hydrogen atoms were
largely unknown. Here, we address this question by comparing fixed geometrically and were not refined. Crystal data are
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reported in the ESI.† The X-ray data of the published structures Synthesis of the complexes
were deposited with the Cambridge Crystallographic Data
Centre CCDC 1495702–1495704.
[Zn(L1)(OAc)](ClO4)2, 1. Zinc(II) perchlorate hexahydrate
(2.00 eq., 744.7 mg, 2.00 mmol) was dissolved in MeOH
(10 cm3) and a methanolic solution (10 cm3) containing HL1
Phosphatase activity measurements
Kinetic measurements of the phosphatase-like activity (1.00 eq., 454.6 mg, 1.00 mmol) and sodium acetate (2.00 eq.,
towards the highly activated substrate bis(2,4-dinitrophenyl) 164.1 mg, 2.00 mmol) was added. The reaction was stirred
phosphate (BDNPP) were carried out by monitoring the for- under reflux for 30 min. After cooling to room temperature, a
mation of 2,4-dinitrophenol (DNP) at 400 nm. A Varian white solid was isolated by filtration and washed with diethyl
Cary50 Bio UV/visible spectrophotometer was employed with ether. The resulting solid was recrystallized in acetonitrile and
10 mm quartz cuvettes and a Peltier temperature controller to colourless crystals (49%, 410.4 mg, 487 µmol) were obtained,
maintain a temperature of 298 K. Measurements were taken which were dried under vacuum. The initial X-ray analysis of a
using a buffer : acetonitrile : DMSO (5 : 4 : 1) solution with a crystal revealed that the cell parameters were identical to those
concentration of complex 1 = 0.5 mM and 2 = 0.25 mM. The previously reported for the complex.67 Elemental analysis:
aqueous buffer consisted of a mixture containing 50 mM Calc. for C29H32Cl2N6O11Zn2: C, 41.35; H, 3.83; N, 9.98%.
MES (2-(N-morpholino)ethanesulfonic acid) (pH 5.5–7.0), Found: C, 41.86; H, 3.92; N, 9.95%. 1H NMR, (δ/ppm, multi-
HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) plicity, J/Hz): 8.59 (2H, d, 4.8), 8.56 (2H, d, 4.8), 8.17–8.02 (4H,
(pH 6.8–8.2), CHES (2-(N-cyclohexylamino)ethane sulfonic m), 7.70–7.51 (8H, m), 4.35–4.19 (6H, m), 4.02 (2H, d, 17.0),
acid) (pH 8.6–10.0) and CAPS (N-cyclohexyl-3-aminopropane- 3.79–3.66 (1H, m), 3.12–3.01 (2H, m), 2.53 (3H, m), 2.04 (2H, t,
sulfonic acid) (pH 9.7–11.1) at constant ionic strength using 11.5). IR: ν = 3630 (w), 3547 (w), 3093 (w), 3039 (w), 2912 (w),
¯
250 mM LiClO4. The pH was adjusted using NaOH to give 2850 (w), 1609 (m), 1576 (m), 1551 (s), 1486 (m), 1457 (m),
buffers ranging from pH 6.0 to 11. These were then treated 1437 (s), 1424 (s), 1076 (s), 1050 (s), 1027 (s), 767 (s), 621 (s)
with Chelex for 24 hours and filtered through a 0.45 μm cm−1
Millex syringe-driven filter. Measurements were carried out as
.
[Zn(HL2)](ClO4)2, 2. An ethanolic (15 cm3) solution contain-
follows: the substrate and the buffer solution were mixed and ing the ligand HL2 was added to an ethanolic solution (5 cm3)
left for one minute before the addition of the complex. Once of Zn(ClO4)2. The white precipitate which formed was filtered
the complex was added the reaction was allowed to run for and washed with diethyl ether. Yield: 0.45 g, 0.72 mmol, 72%.
15 seconds in the measurement chamber for the temperature The solid was recrystallized in hot MeOH, resulting in crystals
to stabilize and the reaction to equilibrate. Then the initial suitable for X-ray diffraction. The IR spectra of the crystalline
rates were determined over a period of three and 10 minutes and the amorphous solids were identical. Elemental analysis:
for complexes 1 and 3, respectively, by recording the emer- Calc. for C21H25Cl2N5O9Zn: C, 40.18; H, 4.01; N, 11.16%.
1
gence of the 2,4-dinitrophenolate product by its strong Found: C, 40.02; H, 4.08; N, 10.83%. H NMR (δ/ppm, multi-
absorption at 400 nm (ε = 12 100 M−1 cm−1);43 measurements plicity, J/Hz): 8.85 (1H, s), 8.29 (1H, t, 7.1), 8.16–8.08 (4H, m),
were carried out in triplicate. The pH dependence of the reac- 7.82–7.78 (2H, m), 7.70 (2H, s), 7.59 (2H, t, 6.4), 5.54–4.96 (2H,
tion was measured over the range of pH 6.0 to 11. The sub- m), 4.33–3.58 (6H, m), 3.21–2.74 (3H, m), 3.79–3.66 (1H, m),
strate concentration dependence at optimum pH was also 3.12–3.01 (2H, m), 2.53 (3H, m), 2.04 (2H, t, 11.5). IR: ν = 3515
¯
measured and the data were fit to the Michaelis–Menten (w), 3235 (w), 3077 (w), 2894 (w), 1615 (m), 1577 (w), 1489 (w),
equation.64
1442 (m), 1071 (s), 1055 (s), 1028 (s), 1010 (s), 768 (s), 758 (s),
619 (s) cm−1
[Zn4(L2)2(OAc)3](ClO4)3·H2O, 3. To a methanolic solution
.
Synthesis of the ligands
The ligands HTPDP (HL1) and HTPPNOL (HL2) (Fig. 1) were (10 cm3) of Zn(ClO4)2 (0.830 g, 2.2 mmol,) a MeOH solution
synthesized as described in the literature.65,66
(20 cm3) containing the ligand HL2 (0.390 g, 1,1 mmol) and
1H NMR (δ/ppm, multiplicity, J/Hz): HL1: 8.49–8.45 (4H, NaOAc (0.180 g, 2.2 mmol) was added. The mixture was
m), 7.54 (4H, td, 1.8), 7.37–7.31 (4H, m), 7.12–7.05 (4H, m), refluxed for 3 h. The reaction was cooled and the solution was
3.98–3.92 (1H, m), 3.81 (8H, s), 2.63 (2H, m), 2.53 (2H, m).13
C
allowed to stand in an open beaker for 48 h. The white solid
NMR (δ/ppm): 159.3, 149.0, 136.5, 123.2, 122.1, 67.2, 60.8, which formed was collected by filtration and washed with
59.1. HL2: 8.52–8.47 (3H, m), 7.66–7.53 (3H, m), 7.35 (1H, d, diethyl ether. Yield: 0.350 g, 0.24 mmol, 44%. The solid was
7.89), 7.27 (2H, d, 7.89), 7.16–7.08 (3H, m), 4.04–4.00(1H, m), recrystallized in hot MeOH, resulting in colourless crystals
3.95 (4H, d, 12.06), 3.85 (2H, d, 14.69), 2.83–2.74 (2H, m), suitable for X-ray diffraction. The IR spectra of the amorphous
2.71–2.64 (2H, m). 13C NMR (δ/ppm): 159.37, 159.08, 149.07, and the crystalline samples were identical. Elemental analysis:
148.85, 136.43, 136.42, 123.05, 122.15, 121.99, 121.86, 67.45, Calc. for C48H59Cl3N10O21Zn4: C, 38.96; H, 4.02; N, 9.46%.
60.42, 59.36, 54.99, 53.15.
Found: C, 38.79; H, 4.06; N, 9.11%. 1H NMR, (δ/ppm, multi-
CAUTION: some of the compounds prepared below are per- plicity, J/Hz): 8.63–8.47 (6H, m), 8.09–7.99 (6H, m), 7.63–7.50
chlorate salts of organic–metal complexes and are potentially (12H, m), 4.45–3.97 (12H, m), 3.93–3.38 (4H, m), 3.08–2.74
explosive. Even small amounts of material should be handled (3H, m), 2.46–2.30 (2H, m), 2.09 (9H, m). IR: ν = 3566 (w), 3324
¯
with caution.
(w), 3238 (w), 3083 (w), 2937 (w), 2862 (w), 1610 (m), 1577 (m),
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1
1554 (s), 1486 (m), 1437 (s), 1408 (m), 1078 (s), 1054 (s), 1026
The complexes were also characterized by H NMR (see ESI
(s), 763 (s), 620 (s) cm−1
.
Fig. S4†). Compound 1 shows a well resolved spectrum in both
aromatic and aliphatic hydrogen regions. Resonances at 2.03
and 3.05 ppm are related to the methylene hydrogen atoms of
the 1,3-diaminopropan-2-ol unit. These hydrogen atoms were
observed as a multiplet at 2.6 ppm in the free ligand. The
methinic hydrogen is observed at 3.72 ppm in the complex
and at 3.9 ppm in the free ligand. The methylene protons
attached to the pyridine groups are observed at 3.98 to
4.35 ppm as a complex multiplet, while in the free ligand they
were observed as a singlet at 3.8 ppm. The resonance at
2.52 ppm, close to the DMSO signal, was attributed to the
methyl hydrogen atoms from the acetate group. Interestingly,
the hydrogen atoms from the acetate group in the compound
Zn(OAc)2(H2O)2 are observed at 1.85 ppm (see ESI Fig. S4†). It
is known that in this compound the acetate is a bidentate
ligand,70 while in 1 it is a bridging ligand. Therefore, this
difference may be ascribed to the different coordination mode
of the acetate group in both compounds.
Results and discussion
Synthesis and characterization of ligands and complexes
HTPDP (HL1) and HTPPNOL (HL2) (Fig. 1) were synthesized
following previously described procedures.65,66 The NMR
spectra of the two ligands (see ESI Fig S1 and S2†) show that
the proposed compounds were obtained in high purity.
Whilst the aromatic hydrogens of the ligand HL1 exhibited
four resonances in the range 7.0–8.5 ppm, in the ligand HL2
these resonances were split into a large number of signals,
and the difference was attributed to the lack of symmetry.
The same effect could be observed in the 13C spectra. The
ligand HL1 exhibited resonances attributed to five aromatic
and three aliphatic carbon atoms whilst the spectrum of the
ligand HL2 shows resonances attributed to ten aromatic and
five aliphatic carbon atoms. Complexes 1 and 3 were prepared
by the reaction of Zn(ClO4)2 in methanol with the appropriate
ligand in the presence of sodium acetate; complex 2 was iso-
lated after the reaction of the ligand HL2 and Zn(ClO4)2 in
ethanol.
The nomenclature employed herein follows schemes
reported previously for these types of ligands and com-
plexes.41,65,66,68,69 This approach denotes the number of remo-
vable protons upon complexation. Both HTPDP (HL1) and
HTPPNOL (HL2) have one potential site for deprotonation;
complexation as L1− and L2− imply the single deprotonation
at the alcohol.
1
The H NMR spectrum of 2 shows the resolution of the res-
onances in the aromatic region. However, the aliphatic hydro-
gen atoms are observed as broad resonances between 2.6 and
5.6 ppm, which indicates a very rigid system. In contrast to the
spectrum observed for 1, no resonance attributed to the
acetate moiety was observed. The 1H NMR spectrum of 3
exhibited lower resolution than the spectra for complexes 1
and 2. The hydrogen atoms from the acetate groups were
observed at 2.09 ppm, different from the chemical shift
observed for Zn(OAc)2(H2O)2 and complex 1.
X-ray crystallography
Crystals of all the three complexes synthesized in this work
were isolated. An initial X-ray analysis of 1 revealed an unit cell
Infrared and NMR spectroscopies
The infrared spectra (see ESI Fig. S3†) of the complexes 1–3 are identical to that already reported,71 in which the ligand HL1
dominated by the intense perchlorate band (1000–1100 cm−1). forms a dinuclear zinc complex, as shown in Fig. 1. The mole-
The IR spectrum of 2 shows the typical CvN and CvC stretch- cular structures solved by X-ray diffraction of compounds 2
ing of the pyridine rings at 1442, 1489, 1577 and 1615 cm−1; 1 and 3 are presented in Fig. 2. Structures containing the ligand
and 3 also show similar bands, but they are overlapped by the HL2 have been published previously for iron and copper
symmetric and asymmetric stretching of the carboxylate derivatives. With iron, a tetranuclear complex was obtained,66
group. For 1 and 3 the υasym of the COO− groups are observed while with copper one dinuclear and two tetranuclear com-
at 1551 and 1554 cm−1, respectively. With respect to the υsym
two intense bands were observed in the range of
,
pounds were reported.72–74
Although the 1,3-diaminopropan-2-ol unit, present in HL2,
1400–1440 cm−1 for both complexes (1424 and 1437 cm−1 for 1 usually results in dinuclear complexes, in the presence of one
and 1408 and 1437 cm−1 for 3). Considering any one of these equivalent of the Zn salt, a mononuclear zinc compound was
bands, the resulting Δ(υasym − υsym) values are in the range formed (Fig. 2). A similar mononuclear species may be formed
observed for bridging carboxylate groups, in agreement with when HL2 reacts with copper,74 but its X-ray molecular struc-
the X-ray molecular structures observed for 1 and 3 (see ture has not yet been reported. The X-ray structure of 2 reveals
below). Compound 2 also shows two low intensity broad the presence of a five-coordinated zinc centre whose coordi-
vibrations at 3235 and 3515 cm−1, which may be attributed to nation environment is composed of the nitrogen donors from
the N–H and O–H stretching; similarly compound 3 displays the ligand HL2. The zinc centre shows a trigonal-bipyramidal
two bands at 3238 and 3324 cm−1 that can be assigned to the geometry (τ = 0.85)75 with a plane formed by nitrogen atoms
N–H vibrations. A secondary amine, as present in complexes 2 from two pyridine groups and one secondary amine. The bond
and 3, should show only one N–H vibration. The presence of lengths between these groups and the zinc centre are very
two such vibrations in 3 may thus indicate the presence of two similar (2.06 Å). In the axial axis, the zinc is coordinated to a
different N–H groups, a suggestion supported by the X-ray pyridine and a tertiary amine that show longer bond distances
structure.
(2.128(2) and 2.197(2) Å, respectively) when compared to the
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ment. Due to the asymmetric coordination of the inter-
dimer acetate bridge the Zn2 site has an N2O4 coordination
environment.
Kinetic studies
Phosphatase-like activities of 1 and 3 were measured using
the activated substrate BDNPP, and the dependence of the
catalytic rate on pH was determined in the pH range 6 to 11
(Fig. 3); 2 was not active under the conditions employed.
Relevant parameters are summarized in Table 1. Previous
studies42,49,50,52,54,57,76 support the underlying assumption
made here that the bridging acetates dissociate in an aqueous
environment, and this dissociation provides the binding sites
for the substrate and water molecules from the solvent.
Compound 3 is very soluble in acetonitrile, in a 1 : 1 mixture
with aqueous buffer a cloudy precipitate appeared. This
problem was overcome when the complex was dissolved in
DMSO instead. However, to avoid precipitation the sequence
of addition of the components differs from the usual
approach, where the substrate is added to the solution (buffer :
acetonitrile) that contains the complex.39,41,42,53 Here, a DMSO
solution of the complexes (0.1 cm3) was added to the buffered
solution containing the substrate (0.5 cm3 buffer, 0.4 cm3 of
acetonitrile). Consequently, the kinetic experiments were per-
formed in a 5 : 4 : 1 buffer : acetonitrile : DMSO mixture. For 1,
the data were collected during the first 180 s where a linear
increase in the absorption was observed. Complex 3, however,
displayed distinctly different behaviour (Fig. 4). After an initial
burst phase lasting ∼60 s the reaction rate gradually leveled
off, reaching a steady-state after ∼120 s. Such a behaviour has
been observed, amongst others, for the peptidase chymotryp-
sin,77 it is reminiscent of the effect of slow binding inhibitors
such as fluoride to PAPs78 and may be associated with a struc-
tural rearrangement induced by the initial interaction between
BDNPP and the catalyst. Therefore, the kinetic data for 3 were
obtained after 120 s.
Fig. 2 X-ray molecular structures of complexes 2 (top) and 3 (bottom).
Hydrogen atoms and counter ions were omitted for clarity. Main bond
lengths for 2 (Å): N(1)–Zn(1) 2.059(2), N(2)–Zn(1) 2.197(2), N(3)–Zn(1)
2.063(2). Main bond lengths for 3 (Å): N(1)–Zn(1) 2.059(6), N(2)–Zn(1)
2.227(5), N(3)–Zn(1) 2.057(6), N(6)–Zn(4) 2.042(6), N(7)–Zn(4) 2.256(6),
N(8)–Zn(4) 2.061(5), Zn(1)–Zn(2) 3.465(5), Zn(3)–Zn(4) 3.484(5), Zn(2)–
Zn(3) 4.106(5). Main bond angles for 2 (°): N(1)–Zn(1)–N(3) 123.09(10),
N(1)–Zn(1)–N(2) 79.74(9), N(3)–Zn(1)–N(2) 79.75(9). Main bond angles
for 3 (°): N(1)–Zn(1)–N(3) 113.7(2), N(1)–Zn(1)–N(2) 78.9(2), N(3)–Zn(1)–
N(2) 79.7(2), N(6)–Zn(4)–N(8) 110.3(2), N(6)–Zn(4)–N(7) 79.1(3), N(8)–
Zn(4)–N(7) 78.1(2).
bonds present in the plane. The alcohol group is not interact-
ing with the zinc centre. The structure shows three five-mem-
bered chelate rings, whose ligand–metal–ligand angles are
close to 80 degrees, and one six-membered ring, with a corres-
ponding angle of 95.9°.
While the reaction of the zinc salt with the hepta-dentate
ligand HL1 in a stoichiometry of 2 : 1 results in the formation
of the dinuclear complex 1, the X-ray analysis of the crystal
obtained for the same reaction conditions, but in the presence
of HL2, revealed a tetranuclear species (Fig. 2) composed of
two dinuclear units (A and B) of the composition Zn2L(OAc);
their inter-dimer Zn–Zn interaction is promoted by an acetate
bridge that acts as a monodentate ligand to one of the Zn(II)
ions but binds in a bidentate manner to the other (coordi-
nation mode μ2:η2–η1), while within one unit the two metals
are bridged by an alkoxide and an acetate group. The metal
ions in the Zn1 and Zn4 positions have the same geometry (tri-
gonal-bipyramidal, τ = 0.8 and 0.9, respectively)75 and coordi-
nation environment (N3O2). The Zn3 site also has a trigonal-
○
1 ( )
Fig. 3 Dependence of phosphatase activity on the pH for
●
and 3 ( ). [BDNPP] = 4.2 mM, [1] = 0.5 mM, [2] = 0.25 mM.
Buffer : acetonitrile : DMSO (5 : 4 : 1) with 50 mM MES, HEPES, CHES and
bipyramidal geometry, but with an N2O3 coordination environ- CAPS, and 250 mM LiClO4, at 25 °C.
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Table 1 Kinetic data and pKa values for 1 and 3
Vmax
Km
(mM)
kcat
kcat/Km
Compound
pKa1
pKa2
(M min−1
)
(min−1
)
(M−1 min−1
)
1
3
7.47
7.72
9.46
9.54
1.57 0.08 × 10−5
3.86 0.6 × 10−6
8.2 0.4
31.4 × 10−3
7.7 × 10−3
3.8
1.8
4.4
1
the residue ascribed to pKa2 may be a terminally coordinated
water. The effect of the substrate concentration on the catalytic
rate of the two complexes was evaluated at optimum pH (i.e.
pH 8.5; Fig. 3). Both data sets display saturation behaviour and
were thus fitted to the Michaelis–Menten equation (Fig. 5 and
Table 1). While complex 1 is approximately four-fold more
reactive than complex 3, its affinity (estimated by the Km value)
for the substrate is reduced. Consequently, 1 is approximately
twice as efficient a catalyst as 3 (as judged by the kcat/Km ratio).
The main difference in the catalytic behaviour of the
two complexes appears to be the number of possible active
species; while for 1 both the singly- and doubly-deproto-
nated species display catalytic activity (with the former
approximately four-fold faster), for 3 only the singly-depro-
tonated species is active. Behaviour similar to that of 1 was
observed previously.41,79–81 In contrast, we are not aware of
other di-zinc(II) complexes that display an initial kinetic
burst as observed for 3 (Fig. 4). Since the initial rate (esti-
mated from the tangent at t = 0 in Fig. 4) during the
burst phase approximates the steady-state rate recorded for
1 under similar experimental conditions, it is possible that
Fig. 4 Plot of absorption vs. time in the reaction of BDNPP with 3 fol-
lowed at 400 nm. Substrate concentrations are indicated on the right,
[3] = 0.25 mM. Buffer : acetonitrile : DMSO (5 : 4 : 1) with 50 mM MES,
HEPES, CHES and CAPS, and 250 mM LiClO4, at 25 °C and pH 8.5. The
inset shows the first 200 s of the reaction progress curve.
a
dinuclear “half” of 3
(i.e. [Zn2(L2)(H2O)n]3+) may be
The pH rate profiles for the two complexes 1 and 3 were
different. While a bell-shaped behaviour is observed for both
with a pH optimum at ∼pH 8.5, for 1 the rate appeared to
approach a limiting rate above pH 11, while 3 is inactive at
high pH. For 1, the data were fitted to eqn (1),64,79
present as the active species; gradual formation of the
tetrameric complex leads to the observed change in the
catalytic rate. Alternatively, the complex may adopt two dis-
tinct conformations, one that is more accessible to the sub-
strate and hence promotes a more rapid turnover, and one
that is more secluded. While the kinetic data do not allow
ꢀ
ꢁ
κKa2
½Hþꢀ
vmax 1 þ
ꢀ
ꢁ
v0 ¼
ð1Þ
½Hþꢀ Ka2
1 þ
þ
Ka1
½Hþꢀ
resulting in pKa1 = 7.47 and pKa2 = 9.46, with κ = 0.2446 being
the ratio of the limiting rate at high pH and the rate at
optimum pH. The data for 3 were fitted to eqn (2) (where
κ = 0),64,79
vmax
ꢀ
ꢁ
v0 ¼
ð2Þ
½Hþꢀ
Ka2
½Hþꢀ
1 þ
þ
Ka1
with pKa1 = 7.72 and pKa2 = 9.54. In both complexes the two
pKas are likely to be associated with Zn(II)-bound water mole-
cules. A plausible interpretation associates pKa1 with the
nucleophilic hydroxide that initiates hydrolysis, while pKa2
may be ascribed to a water molecule that obstructs substrate
coordination due to slow ligand exchange (in its deprotonated
form). The relative acidic value of pKa1, together with its sig-
nificant difference from pKa2 (∼2 pH units) suggests that its
▲
Fig. 5 Substrate dependence measured at pH 8.5 for 0.5 mM 1 ( ) and
■
0.25 mM 3 ( ), buffer : acetonitrile : DMSO (5 : 4 : 1) with 50 mM MES,
associated residue may be a metal ion-bridging water, while HEPES, CHES and CAPS, and 250 mM LiClO4, at 25 °C.
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a
distinction between these two possibilities, crystallo- a complex : reactant ratio of 1 : 1). Under these conditions the
3−
graphic information discussed below favors the latter phosphate ion PO4
displays a resonance at ∼2.7 ppm
mechanism.
(Fig. 6(a)). When complex 1 is added this signal almost dis-
appears but new resonances at 3.50, 3.57, 11.30 and
11.35 ppm emerge (Fig. 6(b)). The resonances around
11.3 ppm account for ∼75% of the phosphate species in solu-
tion, whilst the resonances around 3.5 ppm represent ∼20% of
the phosphate content. The observation of these new reson-
ances after addition of 1 supports the coordination of phos-
phate to the metal ions of the complex. The more moderately
perturbed resonances around 3.5 ppm are, tentatively,
31P NMR studies
In order to probe the details of the catalytic reactions pro-
moted by 1 and 3 further 31P NMR was employed. Initially,
the interactions between 1 and several phosphate species
(i.e. PO43−, 4NPP, B4NPP) were monitored under conditions
similar to those used in the kinetic assays (in
buffer : CD3CN : DMSO-d6 (5 : 4 : 1) mixture at pH 8.5, but with
a
Fig. 6 31P NMR spectra of phosphate (PO43−), 4-nitrophenylphosphate (4NPP) and bis(4-nitrophenyl)phosphate (B4NPP) in the absence and in the
presence of complex 1 at a stoichiometry 1 : 1, at pH 8.5, in CD3CN : buffer : DMSO-d6 (4 : 5 : 1), after 24 h.
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assigned to the coordination of the phosphate ion as a mono- unbound 2,4-BDNPP at −14.10 ppm. After 24 h, a new
dentate ligand to either of the Zn(II); in contrast, the more sig- intense signal (52%) around −0.1 ppm is observed for the
nificantly perturbed resonances around 11.3 ppm are attribu- reaction containing complex 1. From a comparison with the
ted to the coordination of the phosphate ion as a bidentate, data presented above (Fig. 6) and also with the data described
metal ion-bridging ligand. In the case of the monoester 4NPP in the literature,47 this resonance is attributed to the monoe-
alone a resonance at −0.57 ppm is observed, which practically ster 2,4-DNPP. In addition, a resonance at 11.9 ppm is also
disappears after the addition of the complex (Fig. 6(c) and (d)). emerging after 24 h. This signal is close to those observed
However, a new signal that represents 86% of the phosphorus when complex 1 is incubated with the phosphate anion alone
species is observed at −1.16 ppm. It was previously reported (Fig. 6(b)), indicating that 1 is capable of hydrolyzing both
that 4NPP has a resonance at −5.05 ppm in CD3CN that under- the ester bonds of 2,4-BDNPP. Based on the estimated rates
goes a downfield shift to 0.62 ppm after coordination as a brid- of DNPP and phosphate formation (from an analysis of the
ging group (μ2:η1–η1).47 Thus, the considerably smaller shift peak areas of relevant resonances shown in Fig. 7) the hydro-
observed here (from −0.57 to −1.16) may suggest a monoden- lysis of the two ester bonds is likely to occur in a processive
tate coordination mode for this ligand. Lastly, in the case of manner as observed for PAPs (DNPP is less activated than
the diester B4NPP the resonance of the phosphorus nucleus in BDNPP and thus a very poor substrate, preventing accurate
solution is observed at −12.7 ppm (Fig. 6(e)) and does not kinetic measurements under the same experimental con-
change in the presence of complex 1 (Fig. 6(f)), suggesting ditions).82,83 DNPP formation is most rapid in the first
that this reactant does not interact with the zinc ions. This 24 hours of the reaction (its contribution to the NMR reson-
interpretation is in agreement with the observed lack of activity ances increases from 0 to 52.5%) before slowing down signifi-
of both 1 and 3 towards B4NPP.
cantly (with increases of 6.5% and 6% over the following 24
In contrast to B4NPP, 2,4-BDNPP does not only bind to and 72 hours, respectively). In contrast, the rate of phosphate
the metal centre of both complexes, but it is also hydrolyzed. formation remains virtually constant over the entire
Fig. 7 shows the time-dependent 31P NMR spectra of the reac- 120 hours, increasing in species contribution by approxi-
tions between complexes 1 and 3 with 2,4-BDNPP (recorded mately 2% per day.
under stoichiometric conditions). For both complexes the
For the reaction with complex 3 a feature around −0.5 ppm
initial spectra are dominated by the resonance attributed to emerges; after 24 hours of incubation this feature accounts for
Fig. 7 31P NMR spectra at different times of the reactions between complexes 1 (top) and 3 (bottom) with 2,4-BDNPP.
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30% of phosphorus in the sample. This resonance is also N3O2 donor set and the remaining two a N2O3 environment.
likely to be associated with the coordinated monoester 2,4- The tetranuclear species has a centre of inversion. While Zn(1)
DNPP. As more of this product is accumulated the spectral is coordinated by only one oxygen atom from the phosphate
feature is clearly resolved into two distinct bands at ∼−0.3 and moiety, Zn(2) is coordinated by two oxygen atoms located on
∼−0.7 ppm (Fig. 7). A similar splitting of resonances has been two different phosphate groups. Furthermore, while 3 has an
previously observed in phosphate-containing complexes and open structure, complex 4 adopts a more closed configuration,
may be due to the presence of different conformers;47 phos- resembling a parallelogram with Zn⋯Zn distances of 3.612 Å
phorus resonances are influenced by O–P–O angles and the (intradimer) and 5.671 Å (interdimer) (see also Fig. 1). The
torsional angles R–O–P–O(R).84 An additional broad resonance intradimer Zn1⋯Zn2 distance is longer in 4 than in 3 (3.612
appears at −3.75 ppm after several days. While the origin of vs. 3.466 and 3.448 Å), which may be due to different bridging
this resonance is currently obscure it is important to point out groups in the two complexes (phosphate vs. carboxylate). This
that no resonances are observed that may be assigned to the elongated metal–metal distance also results in a more opened
presence of phosphate (as observed for the reaction with 1). alkoxide bridge in 4 in comparison with 3. In the latter, the
Hence, in contrast to 1 complex 3 does not appear to be able Zn–OR–Zn angles are 125.88° and 126.10°, while an angle of
to hydrolyze monoester substrates.
130.87° is observed for 4. The same effect is observed when
complex 1 reacts with PNPP, resulting in the compound
[Zn2(L1)(PNPP)]+; the relevant angle increases from 123° to 129.8°.67
The crystal structure of 4 illustrates that two phosphate
monoesters are tightly coordinated to the zinc centres with an
unusual coordination mode, μ–η1:η1:η1, in which the three
oxygen atoms of DNPP interact with three distinct metal ions.
To our knowledge the only other known example of a phos-
phate complex whereby three oxygen atoms of the ligand
bind to three different metal ions is the compound
[Zn3(CF3SO3)3(μ3-HPO4)L](CF3SO3).85 This tight coordination of
DNPP may account for the lack of phosphate formation by 3
(Fig. 7).
No crystals from the reaction between 1 and BDNPP were
obtained, possibly due to the fact that 1 also hydrolyses DNPP
(Fig. 7). However, the crystal structure of the complex between
1 and the monoester 4NPP was previously reported.67 A dinuc-
lear species was formed, in which the phosphate moiety of
PNPP replaced the bridging acetate.
X-ray structural characterization of the product of the reaction
between 3 and BDNPP
While monitoring the catalytic reaction between 3 and BDNPP
we observed the formation of crystals. Using X-ray diffraction it
could be shown that a tetranuclear species, [Zn4(L2)2(DNPP)2]
(ClO4)2, 4, is formed. Its molecular structure is shown in Fig. 8
and relevant crystallographic parameters, bond lengths and
angles are summarised in Tables S1–S3 (ESI†). The complex
consists of two Zn2L2 subunits, connected via two DNPP mole-
cules. DNPP, as mentioned above, is the product of the hydro-
lysis of BDNPP. The four zinc(II) ions display distorted trigo-
nal-bipyramidal geometry, with two metal ions possessing a
Conclusions
In this study we compared the ligands HL1 (hepta-dentate)
and HL2 (hexa-dentate) with respect to their coordination be-
haviour with zinc(II). While a dinuclear zinc compound was
formed with the former (complex 1), the latter resulted in
either a mononuclear (complex 2) or tetranuclear (complex 3)
species, depending on the amount of metal ions added.
Compound 2 is catalytically inactive, possibly because it does
not provide an adequate binding site for the substrate.
Compound 1 displays the highest steady-state rate (Table 1),
but in the initial burst phase 3 has a rate similar to that of 1
(Fig. 4). Based on the comparison of the crystal structures of
free 3 and product-bound 3 (Fig. 1, 2 and 8) it is likely that the
initial substrate (i.e. BDNPP) binding triggers a structural
change in 3 from a more substrate-accessible to a rather
secluded conformation. This interpretation implies that 3
mimics not only the catalytic activity of metallohydrolases
such as PAPs, but also some of the conformational flexibility
inherent to enzyme-catalysed reactions. With respect to the
catalytic mechanism compounds 1 and 3 are similar, display-
Fig. 8 X-ray molecular structure of complex 4. Hydrogen atoms and
counter ions were omitted for clarity. Main bond lengths for 4 (Å): N(1)–
Zn(1) 2.068(3), N(2)–Zn(1) 2.250(3), N(3)–Zn(1) 2.088(4), Zn(1)–Zn(2)
3.612(3), Zn(1)–Zn(2)i 5.670(3), Zn(1)–Zn(1)i 8.170(3), Zn(2)–Zn(2)i
4.863(3). Main bond angles for 4 (°): N(1)–Zn(1)–N(3) 107.97(13), N(1)–
Zn(1)–N(2) 77.96(13); N(3)–Zn(1)–N(2) 78.74(14).
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ing the maximum activity at ∼pH 8.5. A nucleophile with a pKa
of ∼7.5 (Table 1) is identified as the species initiating hydro-
lysis. A doubly-Lewis activated water molecule bridging two
zinc ions would be a plausible candidate for such a nucleophi-
lic species, an assignment that is in agreement with numerous
studies on metallohydrolase enzymes.1,2 A second catalytically-
relevant pKa (∼9.5; Table 1) is observed for both active com-
pounds. A water molecule coordinated to only one Zn(II) would
be anticipated to possess an acid dissociation constant of this
5 N. Mitić, M. Miraula, C. Selleck, K. S. Hadler,
M. M. Pedroso, E. Uribe and G. Schenk, Adv. Protein Chem.
Struct. Biol., 2014, 97, 49–81.
6 D. M. F. van Aalten, C. R. Chong and L. Joshua-Tor,
Biochemistry, 2000, 39, 10082–10089.
7 D. W. Christianson and W. N. Lipscomb, Acc. Chem. Res.,
1989, 22, 62–69.
8 M. A. Holmes and B. W. Matthews, J. Mol. Biol., 1982, 160,
623–639.
magnitude, as observed in
a
multitude of related
9 O. A. Adekoya and I. Sylte, Chem. Biol. Drug Des., 2009, 73,
7–16.
metallosystems.16,26,28,29,52,86–90 Interestingly, while the depro-
tonation of this water ligand abolishes the catalytic activity of 10 B. W. Matthews, Acc. Chem. Res., 1988, 21, 333–340.
3, compound 1 maintains residual activity at high pH (Fig. 3). 11 J. E. Coleman, Curr. Opin. Chem. Biol., 1998, 2, 222–234.
This may suggest that in 1 the substrate can bind in two 12 B. Krajewska, J. Mol. Catal. B: Enzym., 2009, 59, 9–21.
alternative modes to the metal centre, bridging and terminally 13 E. Jabri, M. B. Carr, R. P. Hausinger and P. A. Karplus,
coordinated, while in 3 only the bridging mode is observed.
Science, 1995, 268, 998–1004.
This difference may also account for the observation that the 14 G. C. Dismukes, Chem. Rev., 1996, 96, 2909–2926.
former promotes the cleavage of both the ester bonds in 15 G. Schenk, N. Mitic, G. R. Hanson and P. Comba, Coord.
BDNPP whereas the latter only hydrolyses one, as observed in
Chem. Rev., 2013, 257, 473–482.
the 31P NMR data (Fig. 7). It is interesting to note that PAP also 16 N. Mitic, K. S. Hadler, L. R. Gahan, A. C. Hengge and
has the ability to cleave both the ester bonds in diester sub-
strates in a processive manner.82
G. Schenk, J. Am. Chem. Soc., 2010, 132, 7049–7054.
17 P. V. Bernhardt, G. Schenk and G. J. Wilson, Biochemistry,
2004, 43, 10387–10392.
In summary, the comparative study of two ligand systems
demonstrated that both may reconstitute features character- 18 J. L. Millan, Purinergic Signalling, 2006, 2, 335–341.
istic of metallohydrolases such as PAPs. Both are catalytically 19 W. Yang, Q. Rev. Biophys., 2011, 44, 1–93.
active towards the diester substrate BDNPP, and while 1 20 Y. Gao, Y. B. Truong, P. Cacioli, P. Butler and I. L. Kyratzis,
mimics the processive hydrolysis of both ester bonds, 3 may
Enzyme Microb. Technol., 2014, 54, 38–44.
demonstrate structural flexibility as observed in its burst kine- 21 F. Ely, K. S. Hadler, N. Mitić, L. R. Gahan, D. L. Ollis,
tics. The next challenge towards building more efficient bio-
mimetics will be combining these features into one system.
Efforts towards this goal are currently in progress.
J. A. Larrabee and G. Schenk, J. Biol. Inorg. Chem., 2011, 16,
777–787.
22 R. Arai, K. Ito, T. Ohnishi, H. Ohba, R. Akasaka, Y. Bessho,
K. Hanawa-Suetsugu, T. Yoshikawa, M. Shirouzu and
S. Yokoyama, Proteins: Struct., Funct., Bioinf., 2007, 67, 732–
742.
Acknowledgements
23 L. J. Daumann, J. A. Larrabee, D. Ollis, G. Schenk and
L. R. Gahan, J. Inorg. Biochem., 2014, 131, 1–7.
The authors acknowledge the financial support received from
Brazilian funding agencies CNPq (406749/2013-2) and CAPES 24 J. Weston, Chem. Rev., 2005, 105, 2151–2174.
(99999.006336/2014-00), as well as a grant from the Australian 25 A. N. Bigley and F. M. Raushel, Biochim. Biophys. Acta,
Research Council (DP150104358). GS also acknowledges the
2013, 1834, 443–453.
Australian Research Council for the award of Future 26 K. S. Hadler, N. Mitic, F. Ely, G. R. Hanson, L. R. Gahan,
a
Fellowship (FT120100694). The authors also acknowledge the
assistance of Prof. Paul V. Bernhardt in collecting the X-ray
diffraction data.
J. A. Larrabee, D. L. Ollis and G. Schenk, J. Am. Chem. Soc.,
2009, 131, 11900–11908.
27 E. Ghanem and F. M. Raushel, Toxicol. Appl. Pharmacol.,
2005, 207, S459–S470.
28 G. Schenk, R. A. Peralta, S. C. Batista, A. J. Bortoluzzi,
B. Szpoganicz, A. K. Dick, P. Herrald, G. R. Hanson,
R. K. Szilagyi, M. J. Riley, L. R. Gahan and A. Neves, J. Biol.
Inorg. Chem., 2008, 13, 139–155.
29 S. J. Smith, R. A. Peralta, R. Jovito, A. Horn, A. J. Bortoluzzi,
C. J. Noble, G. R. Hanson, R. Stranger, V. Jayaratne,
G. Cavigliasso, L. R. Gahan, G. Schenk, O. R. Nascimento,
A. Cavalett, T. Bortolotto, G. Razzera, H. Terenzi, A. Neves
and M. J. Riley, Inorg. Chem., 2012, 51, 2065–2078.
References
1 N. Mitić, S. J. Smith, A. Neves, L. W. Guddat, L. R. Gahan
and G. Schenk, Chem. Rev., 2006, 106, 3338–3363.
2 G. Schenk, N. Mitic, L. R. Gahan, D. L. Ollis, R. P. McGeary
and L. W. Guddat, Acc. Chem. Res., 2012, 45, 1593–1603.
3 M. W. Crowder, J. Spencer and A. J. Vila, Acc. Chem. Res.,
2006, 39, 721–728.
4 G. Schenk, I. Mateen, T.-K. Ng, M. M. Pedroso, N. Mitic, 30 A. Horn Jr., C. Fernandes, G. L. Parrilha, M. M. Kanashiro,
M. Jafelicci, R. F. C. Marques, L. R. Gahan and D. L. Ollis,
F. V. Borges, E. J. T. de Melo, G. Schenk, H. Terenzi and
Coord. Chem. Rev., 2016, 317, 122–131.
C. T. Pich, J. Inorg. Biochem., 2013, 128, 38–47.
Dalton Trans.
This journal is © The Royal Society of Chemistry 2016

View Article Online
Dalton Transactions
Paper
31 A. Horn, I. Vencato, A. J. Bortoluzzi, R. Horner, 51 P. Karsten, A. Neves, A. J. Bortoluzzi, M. Lanznaster and
R. A. N. Silva, B. Spoganicz, V. Drago, H. Terenzi, V. Drago, Inorg. Chem., 2002, 41, 4624–4626.
M. C. B. de Oliveira, R. Werner, W. Haase and A. Neves, 52 F. R. Xavier, A. Neves, A. Casellato, R. A. Peralta,
Inorg. Chim. Acta, 2005, 358, 339–351.
A. J. Bortoluzzi, B. Szpoganicz, P. C. Severino, H. Terenzi,
Z. Tomkowicz, S. Ostrovsky, W. Haase, A. Ozarowski,
J. Krzystek, J. Telser, G. Schenk and L. R. Gahan, Inorg.
Chem., 2009, 48, 7905–7921.
32 B. de Souza, R. Heying, A. J. Bortoluzzi, J. B. Domingos and
A. Neves, J. Mol. Catal. A: Chem., 2015, 397, 76–84.
33 L. Zhang, B. Li, X. Meng, L. Huang and D. Wang, Environ.
Sci. Pollut. Res., 2015, 22, 15104–15112.
34 E. Ghanem, Y. Li, C. Xu and F. M. Raushel, Biochemistry,
2007, 46, 9032–9040.
53 L. J. Daumann, Spectroscopic and Mechanistic Studies
of Dinuclear Metallohydrolases and their Biomimetic
Complexes, Springer International Publishing, Switzerland,
2014.
35 C. Piovezan, R. Jovito, A. J. Bortoluzzi, H. Terenzi,
F. L. Fischer, P. C. Severino, C. T. Pich, G. G. Azzolini, 54 B. Das, H. Daver, A. Singh, R. Singh, M. Haukka,
R. A. Peralta, L. M. Rossi and A. Neves, Inorg. Chem., 2010,
49, 2580–2582.
36 B. de Souza, G. L. Kreft, T. Bortolotto, H. Terenzi,
S. Demeshko, F. Meyer, G. Lisensky, M. Jarenmark,
F. Himo and E. Nordlander, Eur. J. Inorg. Chem., 2014,
2014, 2204–2212.
A. J. Bortoluzzi, E. E. Castellano, R. A. Peralta, J. B. Domingos 55 M. Lanznaster, A. Neves, A. J. Bortoluzzi, B. Szpoganicz and
and A. Neves, Inorg. Chem., 2013, 52, 3594–3596. E. Schwingel, Inorg. Chem., 2002, 41, 5641–5643.
37 A. Neves, M. A. de Brito, I. Vencato, V. Drago, K. Griesar, 56 S. J. Smith, M. J. Riley, C. J. Noble, G. R. Hanson,
W. Haase and Y. P. Mascarenhas, Inorg. Chim. Acta, 1993,
214, 5–8.
R. Stranger, V. Jayaratne, G. Cavigliasso, G. Schenk and
L. R. Gahan, Inorg. Chem., 2009, 48, 10036–10048.
38 A. Neves, M. A. deBrito, I. Vencato, V. Drago, K. Griesar and 57 S. Drueke, K. Wieghardt, B. Nuber, J. Weiss,
W. Haase, Inorg. Chem., 1996, 35, 2360–2368.
39 L. R. Gahan, S. J. Smith, A. Neves and G. Schenk,
Eur. J. Inorg. Chem., 2009, 19, 2745–2758.
40 A. Neves, M. A. de Brito, V. Drago, K. Griesar and W. Haase,
Inorg. Chim. Acta, 1995, 237, 131–135.
41 L. J. Daumann, L. Marty, G. Schenk and L. R. Gahan,
Dalton Trans., 2013, 42, 9574–9584.
42 A. E. Roberts, G. Schenk and L. R. Gahan, Eur. J. Inorg.
Chem., 2015, 2015, 3076–3086.
H. P. Fleischhauer, S. Gehring and W. Haase, J. Am. Chem.
Soc., 1989, 111, 8622–8631.
58 R. Than, A. A. Feldmann and B. Krebs, Coord. Chem. Rev.,
1999, 182, 211–241.
59 A. Neves, S. M. D. Erthal, V. Drago, K. Griesar and
W. Haase, Inorg. Chim. Acta, 1992, 197, 121–124.
60 A. Neves, L. M. Rossi, A. Horn Jr., I. Vencato,
A. J. Bortoluzzi, C. Zucco and A. S. Mangrich, Inorg. Chem.
Commun., 1999, 2, 334–337.
43 R. A. Peralta, A. J. Bortoluzzi, B. de Souza, R. Jovito, 61 L. M. Rossi, A. Neves, A. J. Bortoluzzi, R. Horner,
F. R. Xavier, R. A. Couto, A. Casellato, F. Nome, A. Dick,
L. R. Gahan, G. Schenk, G. R. Hanson, F. C. de Paula,
E. C. Pereira-Maia, P. M. S. de, P. C. Severino, C. Pich,
B. Szpoganicz, H. Terenzi, A. S. Mangrich, E. Pereira-Maia,
E. E. Castellano and W. Haase, Inorg. Chim. Acta, 2005, 358,
1807–1822.
T. Bortolotto, H. Terenzi, E. E. Castellano, A. Neves and 62 G. M. Sheldrick, SHELXL97: Program for the refinement of
M. J. Riley, Inorg. Chem., 2010, 49, 11421–11438. crystal structures, University of Gottingen, Germany, 1997.
44 P. V. Bernhardt, S. Bosch, P. Comba, L. R. Gahan, 63 L. J. Farrugia, J. Appl. Crystallogr., 1999, 32, 837–838.
G. R. Hanson, V. Mereacre, C. J. Noble, A. K. Powell, 64 I. H. Segel, Enzyme kinetics: behavior and analysis of rapid
G. Schenk and H. Wadepohl, Inorg. Chem., 2015, 54, 7249–
equilibrium and steady state enzyme systems, Wiley-
7263.
Interscience, New York, 1975.
45 M. Ghiladi, C. J. McKenzie, A. Meier, A. K. Powell, 65 M. Sato, Y. Mori and T. Iida, Synthesis, 1992, 539–540.
J. Ulstrup and S. Wocadlo, J. Chem. Soc., Dalton Trans., 66 C. Fernandes, A. Neves, I. Vencato, A. J. Bortoluzzi,
1997, 4011–4018.
46 S. Bosch, P. Comba, L. R. Gahan and G. Schenk, J. Inorg.
Biochem., 2016, 162, 343–355.
47 S. Bosch, P. Comba, L. R. Gahan and G. Schenk, Inorg.
Chem., 2014, 53, 9036–9051.
48 P. Comba, L. R. Gahan, G. R. Hanson, V. Mereacre,
C. J. Noble, A. K. Powell, I. Prisecaru, G. Schenk and
V. Drago, T. Weyhermuller and E. Rentschler, Chem. Lett.,
2000, 540–541.
67 E. Kinoshita, M. Takahashi, H. Takeda, M. Shiro and
T. Koike, Dalton Trans., 2004, 1189–1193.
68 L. J. Daumann, K. E. Dalle, G. Schenk, R. P. McGeary,
P. V. Bernhardt, D. L. Ollis and L. R. Gahan, Dalton Trans.,
2012, 41, 1695–1708.
M. Zajaczkowski-Fischer, Chem. – Eur. J., 2012, 18, 1700– 69 L. J. Daumann, P. Comba, J. A. Larrabee, G. Schenk,
1710.
R. Stranger, G. Cavigliasso and L. R. Gahan, Inorg. Chem.,
2013, 52, 2029–2043.
70 J. N. Van Niekerk, F. R. L. Schoening and J. H. Talbot, Acta
Crystallogr., 1953, 6, 720–723.
49 F. R. Xavier, A. J. Bortoluzzi and A. Neves, Chem.
Biodiversity, 2012, 9, 1794–1805.
50 F. R. Xavier, R. A. Peralta, A. J. Bortoluzzi, V. Drago,
E. E. Castellano, W. Haase, Z. Tomkowicz and A. Neves, 71 H. Adams, D. Bradshaw and D. E. Fenton, J. Chem. Soc.,
J. Inorg. Biochem., 2011, 105, 1740–1752.
Dalton Trans., 2002, 925–930.
This journal is © The Royal Society of Chemistry 2016
Dalton Trans.

View Article Online
Paper
Dalton Transactions
72 C. Fernandes, A. Neves, A. J. Bortoluzzi, A. S. Mangrich, 81 K. E. Dalle, L. J. Daumann, G. Schenk, R. P. McGeary,
E. Rentschler, B. Szpoganicz and E. Schwingel, Inorg. Chim.
Acta, 2001, 320, 12–21.
L. R. Hanton and L. R. Gahan, Polyhedron, 2013, 52, 1336–
1343.
73 C. Fernandes, A. J. Bortoluzzi, B. Szpoganicz, E. Schwingel 82 R. S. Cox, G. Schenk, N. Mitić, L. R. Gahan and
and A. Neves, Inorg. Chim. Acta, 2005, 358, 997–1004. A. C. Hengge, J. Am. Chem. Soc., 2007, 129, 9550–9551.
74 C. Fernandes, A. Neves, A. J. Bortoluzzi, B. Szpoganicz 83 K. S. Hadler, L. R. Gahan, D. L. Ollis and G. Schenk,
and E. Schwingel, Inorg. Chem. Commun., 2001, 4,
354–357.
75 A. W. Addison, T. N. Rao, J. Reedijk, J. van Rijn and
J. Inorg. Biochem., 2010, 104, 211–213.
84 D. G. Gorenstein, Phosphorus-31 NMR: Principles and
Applications, Academic Press, 1984.
G. C. Verschoor, J. Chem. Soc., Dalton Trans., 1984, 1349– 85 X. Liu, P. Du and R. Cao, Nat. Commun., 2013, 4, 2375.
1356.
86 F. Ely, K. S. Hadler, L. R. Gahan, L. W. Guddat, D. L. Ollis
76 M. Lanznaster, A. Neves, A. J. Bortoluzzi, V. V. E. Aires,
and G. Schenk, Biochem. J., 2010, 432, 565–573.
B. Szpoganicz, H. Terenz, P. C. Severino, J. M. Fuller, 87 N. Mitic, C. J. Noble, L. R. Gahan, G. R. Hanson and
S. Drew, L. R. Gahan, G. R. Hanson, M. J. Riley and
G. Schenk, J. Biol. Inorg. Chem., 2005, 10, 319–332.
77 T. M. Kitson, Biochim. Biophys. Acta, Protein Struct. Mol.
Enzymol., 1998, 1385, 43–52.
G. Schenk, J. Am. Chem. Soc., 2009, 131, 8173–8179.
88 N. Mitić, M. Valizadeh, E. W. W. Leung, J. de Jersey,
S. Hamilton, D. A. Hume, A. I. Cassady and G. Schenk,
Arch. Biochem. Biophys., 2005, 439, 154–164.
78 T. W. Elliott, N. Mitić, L. R. Gahan, L. W. Guddat and 89 A. Neves, M. Lanznaster, A. J. Bortoluzzi, R. A. Peralta,
G. Schenk, J. Braz. Chem. Soc., 2006, 17, 1558–1565.
79 A. Kantacha, R. Buchholz, S. J. Smith, G. Schenk and
L. R. Gahan, J. Biol. Inorg. Chem., 2011, 16, 25–32.
80 Y. L. M. Zee, L. R. Gahan and G. Schenk, Aust. J. Chem.,
2011, 64, 258–264.
A. Casellato, E. E. Castellano, P. Herrald, M. J. Riley and
G. Schenk, J. Am. Chem. Soc., 2007, 129, 7486–7487.
90 S. J. Smith, A. Casellato, K. S. Hadler, N. Mitic, M. J. Riley,
A. J. Bortoluzzi, B. Szpoganicz, G. Schenk, A. Neves and
L. R. Gahan, J. Biol. Inorg. Chem., 2007, 12, 1207–1220.
Dalton Trans.
This journal is © The Royal Society of Chemistry 2016